1. Field of the Invention
This invention relates to a human hematopoietic growth regulatory gene termed hiwi and genes corresponding thereto. Specifically, the invention relates to the isolation, cloning and sequencing of complementary DNA (cDNA) copies of messenger RNA (mRNA) encoding a novel human hiwi gene. The invention also relates to the construction of recombinant expression constructs comprising cDNA of the novel human hiwi gene, said recombinant expression constructs being capable of expressing hiwi gene product in cultures of transformed prokaryotic and eukaryotic cells. Production of the hiwi gene product in such cultures is also provided. The invention relates to the use of such cultures of such transformed cells to produce homogeneous compositions of the human hiwi gene product. The invention also provides cultures of such cells producing the hiwi protein for the characterization of novel and useful drugs. Antibodies against and epitopes of this novel human hiwi gene product are also provided by the invention. Methods for isolating human hematopoietic stem cells from biological samples such as bone marrow are also provided.
2. Background of the Invention
Stem cells can undergo self-renewal as well as generate differentiated progeny. Hematopoietic stem cells (HSC) have the ability to undergo self-renewal and to differentiate into cells belonging to multiple hematopoietic lineages (Morrison et al., 1995, Annu Rev Cell Dev Biol. 11:35-71; Chen et al., 1997, Immunol Rev. 157:41-51). The capacity of a hematopoietic stem cell to remain undifferentiated and be capable of reconstituting a myeloablated host as well as its ability to generate multiple differentiated cell types is central to its pivotal role in normal hematopoiesis. These properties allow stem cells to maintain hematopoiesis throughout the lifespan of an organism.
The knowledge of the behavior of HSCs is limited due to their rarity, difficulty of efficient isolation, and their sensitivity to manipulation (Morrison et al., ibid.; Chen et al., ibid.). Despite an improved ability of various laboratories to isolate and manipulate pure populations of murine and human HSCs (Goodell, 1999, Blood 94:2545-2547; Huang et al., 1999, Blood 94:2595-2604) current understanding of mechanisms by which a stem cell divides and retains its unique biological properties has eluded the efforts of a large number of investigators (Vaziri et al., 1994, Proc Natl. Acad Sci. USA 91:9857-9860; Lansdorp et al., 1995, Exp Hematol. 23:187-191; van der Loo et al., 1995, Blood 85:2598-2606; Peters et al., 1995, Exp Hematol. 23:461-469; Peters et al., 1996, Blood 87:30-37; Yonemura et al., 1996, Proc Natl Acad Sci. USA 93:4040-4044).
Elucidation of the genetic program that underlies the unique biological properties of HSCs has been the focus of a growing number of laboratory groups (Vaziri et al., ibid.; Lansdorp et al., ibid.) using a variety of approaches. Array technology, for instance, now permits simultaneous monitoring of expression patterns of thousands of genes during cellular differentiation and response (van der Loo et al., ibid.; Peters et al., ibid.). The key to the successful implementation of such technology to the study of stem cell biology is the development of the means to assign priority to such genes and to determine their function.
The self-renewal capacity of several classes of stem cells is thought to be controlled by external signals and intrinsic cellular processes (Morrison et al., ibid.; Chen et al., ibid.; Bruno et al., 1995, Exp Hematol. 23:1212-1217; Hoffman, 1999, Curr Opin Hematol. 6:184-191). Over the last 2 decades, a variety of external stimuli (cytokines, matrix proteins) that alter HSC self-renewal have been the subject of intense investigation. Although a number of such external signals that interact with specific receptors on HSC have been identified, the signaling mechanisms that govern HSC self-renewal have eluded investigation.
A different approach to analyze the genetic organization of human HSCs is to analyze expression of genes originally shown to affect stem cell development in lower species (Peters et al., ibid.; Yonemura et al., ibid.; Zon 1995, Blood 86:2876-2891). In these experiments, genes that were originally shown to affect stem cell development in lower species have been shown subsequently to be expressed by human hematopoietic cells and to have profound regulatory effect on human hematopoiesis. Lower organisms such as Drosophilae, C. elegans and D. rerio (zebra fish) have been utilized as effective models for studying mechanisms that are conserved among diverse developmental systems (Lewis, 1978, Nature 276:565-570; Zon, 1995, ibid.; Tabara et al., 1999, Cell 99:123-132). Studies from Xenopus, for instance, have revealed a multitude of genes involved in mesoderm induction including members of the transforming growth factor β superfamily, fibroblast growth factor and at least 19 members of the Wnt gene family have been identified in diverse species ranging from roundworm and insects to humans (Sidow, 1992, Proc Natl Acad Sci. USA 89:5098-5102; Austin et al., 1997, Blood 89:3624-3635). Wnt gene family members have subsequently been shown to have profound effects on murine and human hematopoiesis.
Intrinsic cellular mechanisms that regulate stem cell self-renewal have been explored in a variety of model systems including germ line stem cells (GSCs) in several lower species. Drosophila has been a particularly useful model for studying biological processes that are conserved in higher developmental systems (Lewis, ibid.; Nusslein-Volhard et al., 1980, Nature 287:795-801; Lin & Spradling, 1993, Dev Biol. 159:140-152; Lin et al., 1997, Development 124:2463-2476; Lin, 1998, Curr Opin Cell Biol. 10:687-693; Cox et al., 1998, Genes Dev. 12:3715-3727; Lin, 1999, Annu Rev Genet. 31:455-491; Benfey, 1999, Curr Biol. R171). In Drosophila, stem cells exist in the germ line at the apical tip of each ovariole, the germarium, which is the functional portion of the ovary (Lin & Spradling, ibid.; King, 1970, OVARIAN DEVELOPMENT IN DROSOPHILA MELANOGASTER,” New York: McGraw-Hill). Each ovary consists of 10-17 ovarioles. Each germarium contains 2 to 3 GSCs that are in direct contact with specialized somatic cells, the basal terminal filament cells (King, 1970, ibid.; Lin, 1998, ibid; Lin, 1999, ibid.). GSCs undergo asymmetric divisions to produce daughter stem cells and a differentiated daughter cell, a cystoblast. The GSCs provide a continuous source of totipotent cells for the production of gametes needed for fertilization (Lin, 1999, ibid.). They are very similar to HSCs in their ability to not only self-renew but to remain capable of generating large numbers of differentiated daughter cells (Lin, 1999, ibid.; Benfey, 1999, ibid.). The intracellular mechanisms which serve as the determinants of asymmetric-segregating cell fates of GSCs depend not only on the basic cell cycle machinery but also on a family of recently identified genes, some of which are evolutionarily conserved (Cox et al., 1998, ibid.; Benfey, 1999, ibid,). A group of somatic cells in Drosophila, termed terminal filament cells, which are distal and immediately adjacent to the GSCs, have been shown to regulate GSC division (Lin, 1998, ibid.; Lin, 1999, ibid.; Lin & Spradling, ibid). Laser ablation of the terminal filament increases the rate of oogenesis by 40% (Lin & Spradling, ibid.).
Recently a number of genes including dpp, piwi, pumilio and fs(1)Yb have been identified and shown to be essential for GSC maintenance (Lin & Spradling, ibid.; Cox et al., 1998, ibid.; King & Lin, 1999, Development 126:1833-1844). Among these genes, piwi has been of special interest. It has recently has been demonstrated to be an essential stem cell gene in Drosophila and C. elegans and to be expressed in tissues belonging to many species including human. The Drosophila piwi gene is required for asymmetric division of GSCs but is not required for differentiation of committed daughter cells. piwi expression in adjacent somatic cells, terminal filament cells, regulates GSC division (Cox et al., 1998, ibid.). Loss-of-function mutations in the piwi gene found in the terminal filament leads to a failure of stem cell maintenanc3 (Lin & Spradling, ibid.; Cox et al., 1998, ibid.); piwi is expressed not only in the terminal filament but also in the germ line. Loss of piwi function in the germ line, however, is not known to affect GSC division. The protein encoded by piwi is extraordinarily well conserved along the evolutionary tree, being found in both Caenorhabditis elegans and primates (Cox et al., 1998, ibid).
Thus, there is a need in the art to identify genes and gene products in hematopoietic stem cells that regulate cell cycling and proliferation. There further is a need in the art to identify a human homolog for the Drosophila piwi gene to determine the role of said homolog in hematopoietic stem cell development and maintenance. There is also a need in the art to develop drugs and other active agents for controlling, promoting or inhibiting hematopoietic stem cell growth, proliferation and differentiation to permit manipulation of hematopoietic stem cells and provide renewable sources of said stem cells. There is additionally a need for developing compounds to inhibit leukemia cell growth and induce apoptosis of such cells as a means of cancer treatment.